Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels.
Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels.
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DOI：
10.1085/jgp.201411347
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发表时间：
2015-04
期刊：
影响因子：
--
通讯作者：
Gadsby DC
中科院分区：
文献类型：
--
作者：
Chaves LA;Gadsby DC
在CFTR通道关闭过程中，催化活性和非活性界面ATP结合位点都至少开放8个碱基。囊性纤维化跨膜传导调节因子（CFTR）通道的打开和关闭由三磷酸腺苷（ATP）结合诱导的形成和水解触发的其胞质核苷酸结合结构域（NBD）的异二聚体的破坏的循环驱动。虽然封闭在异二聚体界面内的两个复合位点在开放的CFTR通道中含有ATP，但在唯一催化活性位点中的ATP水解导致通道关闭。打开该站点的NBD接口即可进行ADP-ATP交换。但是，NBD表面在另一个不活跃的复合地点分离的频率和距离仍不清楚。我们通过监测核苷酸大小的亲水性、硫醇特异性甲硫基磺酸盐（MTS）试剂与引入LSGGQ样ATP结合盒特征序列（替换等效保守丝氨酸：S549和S1347）的界面靶半胱氨酸的接触，评估了每个复合位点的分离。共价MTS依赖性修饰的半胱氨酸，而通道保持关闭的情况下，ATP受损的后续开放后，ATP readdition。当通道在ATP存在下打开和关闭时的修饰引起宏观CFTR电流以与当未修饰的通道在突然ATP撤回时关闭时相同的速度下降。这些结果表明，目标半胱氨酸只能在封闭的通道中被修饰;修饰后，附着的MTS加合物干扰ATP介导的开放;一旦通道关闭，在它们重新打开之前，在ATP存在下的修饰迅速发生。这一解释得到以下发现的证实：对于任一半胱氨酸靶点，添加水解损害突变K1250 R（催化位点步行者A Lys）同样使ATP去除时通道关闭和MTS试剂修饰ATP的速度减慢一个数量级。我们的结论是，在每个CFTR通道门控循环，NBD二聚体界面分离同时在两个复合网站足以让MTS试剂访问两个签名序列丝氨酸。用较大的试剂MTS-葡萄糖、MTS-生物素和MTS-罗丹明对S1347 C通道进行相对快速的修饰表明，在非催化复合位点，这种分离必须超过8 μ m。
Both the catalytically active and inactive interfacial ATP-binding sites open at least 8 Å during CFTR channel closure. Cystic fibrosis transmembrane conductance regulator (CFTR) channel opening and closing are driven by cycles of adenosine triphosphate (ATP) binding–induced formation and hydrolysis-triggered disruption of a heterodimer of its cytoplasmic nucleotide-binding domains (NBDs). Although both composite sites enclosed within the heterodimer interface contain ATP in an open CFTR channel, ATP hydrolysis in the sole catalytically competent site causes channel closure. Opening of the NBD interface at that site then allows ADP–ATP exchange. But how frequently, and how far, the NBD surfaces separate at the other, inactive composite site remains unclear. We assessed separation at each composite site by monitoring access of nucleotide-sized hydrophilic, thiol-specific methanothiosulfonate (MTS) reagents to interfacial target cysteines introduced into either LSGGQ-like ATP-binding cassette signature sequence (replacing equivalent conserved serines: S549 and S1347). Covalent MTS-dependent modification of either cysteine while channels were kept closed by the absence of ATP impaired subsequent opening upon ATP readdition. Modification while channels were opening and closing in the presence of ATP caused macroscopic CFTR current to decline at the same speed as when the unmodified channels shut upon sudden ATP withdrawal. These results suggest that the target cysteines can be modified only in closed channels; that after modification the attached MTS adduct interferes with ATP-mediated opening; and that modification in the presence of ATP occurs rapidly once channels close, before they can reopen. This interpretation was corroborated by the finding that, for either cysteine target, the addition of the hydrolysis-impairing mutation K1250R (catalytic site Walker A Lys) similarly slowed, by an order of magnitude, channel closing on ATP removal and the speed of modification by MTS reagent in ATP. We conclude that, in every CFTR channel gating cycle, the NBD dimer interface separates simultaneously at both composite sites sufficiently to allow MTS reagents to access both signature-sequence serines. Relatively rapid modification of S1347C channels by larger reagents—MTS-glucose, MTS-biotin, and MTS-rhodamine—demonstrates that, at the noncatalytic composite site, this separation must exceed 8 Å.
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影响因子：
5.5
作者：
Cui, L;Aleksandrov, L;Aleksandrov, AA
通讯作者：
Aleksandrov, AA
影响因子：
16.8
作者：
Boël G;Smith PC;Ning W;Englander MT;Chen B;Hashem Y;Testa AJ;Fischer JJ;Wieden HJ;Frank J;Gonzalez RL Jr;Hunt JF
通讯作者：
Hunt JF
DOI：
10.1073/pnas.0911061107
发表时间：
2010-01-19
影响因子：
11.1
作者：
Csanady, Laszlo;Vergani, Paola;Gadsby, David C.
通讯作者：
Gadsby, David C.
DOI：
10.1126/science.1168750
发表时间：
2009-03-27
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Aller SG;Yu J;Ward A;Weng Y;Chittaboina S;Zhuo R;Harrell PM;Trinh YT;Zhang Q;Urbatsch IL;Chang G
通讯作者：
Chang G
DOI：
10.1085/jgp.200308798
发表时间：
2003-09
期刊：
The Journal of general physiology
影响因子：
--
作者：
Basso C;Vergani P;Nairn AC;Gadsby DC
通讯作者：
Gadsby DC